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BACKGROUND: Slow and cumbersome laboratory diagnostics for Mycobacterium tuberculosis complex (MTBC) risk delayed treatment and poor patient outcomes. Whole-genome sequencing (WGS) could potentially provide a rapid and comprehensive diagnostic solution. In this prospective study, we compare real-time WGS with routine MTBC diagnostic workflows. METHODS: We compared sequencing mycobacteria from all newly positive liquid cultures with routine laboratory diagnostic workflows across eight laboratories in Europe and North America for diagnostic accuracy, processing times, and cost between Sept 6, 2013, and April 14, 2014. We sequenced specimens once using local Illumina MiSeq platforms and processed data centrally using a semi-automated bioinformatics pipeline. We identified species or complex using gene presence or absence, predicted drug susceptibilities from resistance-conferring mutations identified from reference-mapped MTBC genomes, and calculated genetic distance to previously sequenced UK MTBC isolates to detect outbreaks. WGS data processing and analysis was done by staff masked to routine reference laboratory and clinical results. We also did a microcosting analysis to assess the financial viability of WGS-based diagnostics. FINDINGS: Compared with routine results, WGS predicted species with 93% (95% CI 90-96; 322 of 345 specimens; 356 mycobacteria specimens submitted) accuracy and drug susceptibility also with 93% (91-95; 628 of 672 specimens; 168 MTBC specimens identified) accuracy, with one sequencing attempt. WGS linked 15 (16% [95% CI 10-26]) of 91 UK patients to an outbreak. WGS diagnosed a case of multidrug-resistant tuberculosis before routine diagnosis was completed and discovered a new multidrug-resistant tuberculosis cluster. Full WGS diagnostics could be generated in a median of 9 days (IQR 6-10), a median of 21 days (IQR 14-32) faster than final reference laboratory reports were produced (median of 31 days [IQR 21-44]), at a cost of £481 per culture-positive specimen, whereas routine diagnosis costs £518, equating to a WGS-based diagnosis cost that is 7% cheaper annually than are present diagnostic workflows. INTERPRETATION: We have shown that WGS has a scalable, rapid turnaround, and is a financially feasible method for full MTBC diagnostics. Continued improvements to mycobacterial processing, bioinformatics, and analysis will improve the accuracy, speed, and scope of WGS-based diagnosis. FUNDING: National Institute for Health Research, Department of Health, Wellcome Trust, British Colombia Centre for Disease Control Foundation for Population and Public Health, Department of Clinical Microbiology, Trinity College Dublin.